Electroacupuncture treatment partly promotes the recovery time of postoperative ileus by activating the vagus nerve but not regulating local inflammation

Electroacupuncture treatment partly promotes the recovery time of postoperative ileus by activating the vagus nerve but not regulating local inflammation 1Scientific RepoRts | 7 39801 | DOI 10 1038/sr[.]

partly promotes the recovery time

of postoperative ileus by activating the vagus nerve but not regulating local inflammation

Jun-fan Fang, Jian-qiao Fang, Xiao-mei Shao, Jun-ying Du, Yi Liang, Wen Wang & Zhe Liu Postoperative ileus (POI) after abdominal surgery significantly lowers the life quality of patients and increase hospital costs However, few treatment strategies have successfully shortened the duration of POI Electroacupuncture (EA) is a modern way of administering acupuncture and widely used in various gastrointestinal (GI) diseases in the world Here, we studied the effect of EA on POI and its underlying mechanisms Intestinal manipulation resulted in significant delays of GI transit, colonic transit and gastric emptying Surgery also up-regulated c-fos in nucleus of the solitary tract (NTS) and induced inflammation response in the small intestine Further, operation and inhale anesthesia inhibited NTS neuron excitation duration for the whole observation time EA administered at ST36 indeed shortened the recovery time of GI and colonic transit, and significantly increased the gastric emptying EA also significantly activated the NTS neurons after operation However, there was no anti-inflammation effect of EA during the whole experiment Finally, atropine blocked the regulatory effect of EA on GI function, when it was injected after surgery, but not before surgery Thus, the regulatory effect of EA

on POI was mainly mediated by exciting NTS neurons to improve the GI tract transit function but not by activating cholinergic anti-inflammatory pathway.

Abdominal and extra-abdominal surgery can lead to impaired motility of the entire gastrointestinal (GI) tract, which is referred to as postoperative ileus(POI)1 Depending on the type of surgery, POI may last over 2 weeks, with symptoms including nausea, vomiting, intolerance to food, absence of defecation, longer hospital stay2 Therefore, POI significantly increases the hospitalization costs

It is becoming increasingly clear that POI is associated with a localized inflammatory reaction, which is a complex orchestrated immune response to intestinal manipulation (IM)1,3 IM induced the influx of leukocytes, mainly monocytes, into the muscularis, after surgery4–6 Secretion of cytokines (IL-6, TNF-α ) from monocytes and smooth muscle inhibitory substances (NO, COX-2) contribute to the delay in intestinal transit7,8 These events delay GI transit, decrease local neuromuscular function and inhibit neurogenic pathways, thereby sup-pressing motility along the entire gastrointestinal tract for sustained postoperative periods9,10

Many new therapeutic strategies for POI has been introduced in the last decade, but with limited success11 Local inflammatory responses during POI are subject to regulation by the vagus nerve system12,13 Electrical stimulation of the vagus efferent nerve or drug stimulation of the nucleus of the solitary tract (NTS), the parasym-pathetic preganglionic center, reduces the inflammatory response and POI via the cholinergic anti-inflammatory pathway14,15 However, for medical ethics and safety reasons, these stimulation methods have been tried in ani-mals or a few patient volunteers

Electroacupuncture (EA), which is a modern way of administering acupuncture, refers to the application of a pulsating electrical current to acupuncture needles for acupoint stimulation It has been widely used for various

GI discords in China and the West16–18 EA promotes the GI function and some reports have shown therapeutic

Department of Neurobiology & Acupuncture Research, the Third Clinical College, Zhejiang Chinese Medical University, Hangzhou, China Correspondence and requests for materials should be addressed to J.-q.F (email: fangjianqiao7532@163.com)

Received: 15 June 2016

Accepted: 29 November 2016

Published: 04 January 2017

effects of EA on POI19,20 Furthermore, EA increase the motility of GI by regulating the vagus activation21 If

EA activated vagus nerve system before surgery, it might prevent the POI by the cholinergic anti-inflammatory pathway such as previous study Here, we focused on the regulatory effect of EA on GI motility after abdominal surgery and investigated the role of vagus nerve in EA’s regulatory effect on POI

Results

GI transit The distribution and geometric center (GC) of fluorescent markers are shown in Fig. 1 In the control group, the fluorescein isothiocyanate-labeled dextran (FITC-dextran) reached the seventh segment of the small bowel (sb-7) as reflected by a GC of 3.38 ± 0.37 at 6 h after surgical manipulation (Fig. 1A) The marker in the model group, EA group and sham EA group remained in the stomach and were distributed mainly

through-out sb-3, as reflected by respective GC values of 1.86 ± 0.24, 1.45 ± 0.15, 1.63 ± 0.16 (P < 0.01) There was no significant difference between the model, EA and sham EA groups (P > 0.05) (Fig. 1B) Similarly, IM

signifi-cantly delayed the GI transit of the model group, EA group and sham EA group (GC = 1.77 ± 0.24, 1.63 ± 0.26,

1.95 ± 0.22, respectively) compared with control 12 h after operation (GC = 5.44 ± 0.37, P < 0.01) EA had no significant therapeutic effect on gastrointestinal motility at this time point (P > 0.05) (Fig. 1C/D) Relative to

the control group (GC = 6.38 ± 0.32), the transit distribution histograms demonstrated a significant delay of GI

transit in the model, EA and sham EA groups (CG = 2.49 ± 0.24, 3.54 ± 0.25, 2.5 ± 0.34, respectively, P < 0.01) at

24 h after surgery However, the fluorescent marker remaining in the stomach of the EA group was less than that

of the model and sham EA groups, with a distributed to sb-6 (Fig. 1E) Although the GCs of model group, EA group and sham EA group were lower than control, the EA group’s GC was much higher than those of the model

group and sham EA group (Fig. 1F, P < 0.05) Throughout the experiment, sham EA did not change GI transit compared with the model group (P > 0.05) The above data indicate that IM caused a clinically relevant POI and

EA treatment partly mitigated the delay of GI transit up to 24 h after surgery

Colonic transit The mean colonic transit times in all experimental groups at each time point are shown in

Fig. 2A The repeated-measures ANOVA with between-subject factors revealed differences over time (P < 0.01) and between groups (P < 0.01) There were significant interactive effects between time points and groups (P < 0.01) Post-hoc LSD tests indicated that IM caused a delay of colonic transit in rats (P < 0.01) A significant difference of colonic transit time was observed in the EA group compared with the model group (P < 0.01)

However, sham EA stimulation did not change the colonic transit time of rats compared with the model group

(P > 0.05).

Comparing colonic transit time between groups at each time point, significant differences occurred among

the control, model, EA and sham EA groups from 6 h to 24 h after IM (P < 0.01) Compared with the control

group, IM significantly delayed the colonic transit of the model and sham EA groups during the observation time

(P < 0.01) However, IM only significantly increased the EA group’s colonic transit time only 12 h after surgery (P < 0.01) Following the EA treatment, the colonic transit time was significantly decreased compared the model

Figure 1 Gastrointestinal tract transit after abdomen operation Manipulation of the rat intestine leads

to a postoperative delay of intestinal transit as measured by the distribution and geometric center (GC) of fluorescent marker and EA partly recovered the gastrointestinal transit at 24 h after operation The distribution

of orally administered FITC-dextran in whole gastrointestinal tract 6 h (A), 12 h (C) and 24 h (E) after operation and the mean GC of fluorescent marker 6 h (B), 12 h (D) and 24 h (F) after surgery n = 6–8 rats per group and

data are means ± SEM **P < 0.01 versus control group, ΔP < 0.05 versus model group, #P < 0.05 versus EA

group

and sham EA groups 6 h after surgery (P < 0.05) Furthermore, 24 h after IM, the colonic transit function of the

EA group recovered to normal level (P > 0.05).

Gastric emptying The mean gastric emptying levels in all experimental groups at 24 h after operation are shown in Fig. 2B Manipulation of the small intestine initiated a significant increase in gastric retention 24 h

after surgery compared with control (P < 0.01) Compared with the model group, EA treatment significantly decreased the gastric retention 24 h after IM (P < 0.01), but sham EA stimulation did not change the gastric func-tion (P > 0.05) However, there was still a significant difference between the EA group and the control group 24 h after IM (P < 0.01).

Expression of c-fos in NTS To obtain more insights into the central mechanism by which EA regulates POI, immunofluorescence analysis of the brain for c-fos was performed The expression of c-fos in the NTS at 6 h,

12 h and 24 h after IM is shown in Fig. 3 High expression of c-fos in the NTS were found at 24 h after surgery in all rats that underwent abdominal operation (Fig. 3A–D) Quantitative c-fos analysis 6 h, 12 h and 24 h after IM showed great number of c-fos-positive neurons in the model group, EA group and sham EA group compared with

the control group (P < 0.01, Fig. 3E–G) EA and sham EA stimulation did not produce any regulatory effect on c-fos expression at 6 h, 12 h and 24 h after surgery compared with the model group (P > 0.05, Fig. 3E–G).

NTS exciting in POI rats We next investigated the effect of EA on the NTS of POI rats by in vivo

elec-trophysiology One hundred sixty-six cingulate neurons from 20 rats were recorded Forty-three were from the control group, 49 were from the model group, 43 were from the EA group and 41 from the sham EA group The number of three type neurons (excited, inhibited, and no response) and the rate of change of spike frequency are shown in Fig. 4

More than 50% of the NTS neurons of rats in the control group were inhibited immediately after the sur-gery (Fig. 4A) These results indicate that only isoflurane, without small bowel manipulation, down-regulated the NTS neuron excitability, which was consistent with the inhibitory effect of isoflurane on GI However, the effect of isoflurane faded by 6 h after the surgery, and the numbers of the three types NTS neurons in the control group tended to equilibrium (Fig. 4C) As with GI function, small bowel manipulation significantly inhibited the NTS neuronal excitability and decreased the spike frequency when compared with the control group during the

whole observation time (P < 0.01, Fig. 4A–H) EA and sham EA stimulation did not produce any significantly

effect on NTS neuronal excitability or spike frequency compared with the model group immediately after

oper-ation (P > 0.05, Fig. 4A,B) However, EA stimuloper-ation significantly up-regulated the NTS neuronal excitability and increased the spike frequency compared with the control and model groups at 6 h after operation (P < 0.05,

Fig. 4C,D) Similarly, from 12 h to 24 h, EA significantly excited the NTS neurons and increased the spike firing

frequency compared with the model group (P < 0.01, Fig. 4E,H) However, no distinct difference between the EA and control groups was observed at the above time points (P > 0.05, Fig. 4E,H).

NTS excitation in normal rats To investigate the effect of 5 Hz EA stimulation at ST36 on NTS neurons before surgery, we studied EA’s effect on normal rats One hundred thirty-two cingulate neurons from 10 rats were recorded Sixty-three were from the control group, and 69 were from the EA group The numbers of the three types of neurons and the rate of change of spike frequency are shown in Fig. 5 Throughout the observation time, more than 50% of the neurons showed no response in the normal group (Fig. 5A,C,E,G,I), which indicated that the NTS neuronal excitability tend to stabilize without stimulation (Fig. 5B,D,F,H,J) After 30 min of 5 Hz

EA stimulation, more than 50% of the neurons in the NTS were excited (Fig. 5A,C,E,G,I), and the NTS neuronal

Figure 2 Colonic transit and gastric emptying after abdomen operation Surgery manipulation of the

rat small intestine produced a pronounced increase of colonic transit time of a 5-mm stainless steel ball and

a significantly increase of gastric retention EA stimulation at ST36 reversed them The mean colonic transit

time of a stainless steel ball was calculated 6 h, 12 h and 24 h after surgery (A) and the average gastric retention

24 h after operation (B) n = 6–8 rats per group and data are means ± SEM **P < 0.01 versus control group,

ΔΔP < 0.01 versus model group, ##P < 0.01 versus EA group.

excitability and spike frequency were significantly increased (P < 0.05, Fig. 5B,D,F,H,J) These findings indicate

that 5 Hz EA simulation at ST 36 produced a persistent excitation on NTS neurons that lasted at least 10 min

Leukocyte infiltration into the intestinal wall Myeloperoxidase (MPO) staining at each time point is shown in Fig. 6 IM induced a significant increase in the number of MPO-positive cells in the intestinal wall 6 h

after IM compared with laparotomy alone (P < 0.01, Fig. 6E) This leukocyte recruitment was not regulated by EA

or sham EA stimulation compared with the model group (P > 0.05, Fig. 6E) Furthermore, the influx of inflam-matory cells in the model group was even more pronounced at 12 h (Fig. 6A–D) and 24 h after surgery (P < 0.01,

Fig. 6F,G) Most importantly, no anti-inflammatory effect of EA treatment was observed during the experiment compared with the model and sham EA groups

Expression of IL-1β and TNF-α mRNA Along with the leukocyte recruitment to the intestinal wall from

6 h to 24 h after surgery, we chose this period to test the effect of EA on some proinflammatory gene expres-sion There were no differences in the expression of IL-1β and TNF-α mRNA expression at 6 h after surgery

(P > 0.05, Fig. 7A,D) However, intestinal manipulation up-regulated the IL-1β mRNA expression from 12 h to

24 h after surgery compared with control (P < 0.01, Fig. 7B,C) IM also increased TNF-α mRNA 24 h after surgery (P < 0.01, Fig. 7F) The expression of IL-1β and TNF-α in the EA group showed no significant difference from either the model group or the sham EA group for the full observation time (P > 0.05, Fig. 7A–F).

The atropine anti-EA regulation effect The above results indicated that EA may promote GI transit function through the vagus nerve, so we injected atropine intraperitoneally before EA administration As shown

in Fig. 8A,B, the distribution of marker in the atropine plus EA group was different from the model group and EA plus atropine group, and the GC of the atropine plus EA group was much higher than those of model group and

EA plus atropine group (P < 0.05) There was no significant difference between the model and EA plus atropine group (P > 0.05) Gastric emptying and colonic transit testing showed similar results (Fig. 8C,D) EA simulation

significantly decreased the relative gastric content and colonic transit time of POI rats when the atropine was

injected before operation (P < 0.01) However, atropine given after surgery inhibited EA enhancing effect on gas-tric emptying and colonic transit (P > 0.05) Furthermore, administration atropine significantly delayed the colon transit of rats (P < 0.01) We found the stainless steel ball in the colon when the rats were sacrificed.

Figure 3 c-fos expression in nucleus of solitary tract after abdomen operation Manipulation of the

gastrointestinal tract promoted the c-fos expression level of bilateral nucleus of solitary tract (NTS), however,

EA simulation produced little effect on these change Immunofluorescence staining of c-fos expression at

bilateral NTS from rats that underwent a laparotomy alone (A), a laparotomy with intestinal manipulation (B), a intestinal manipulation with EA stimulation (C) and intestinal manipulation with sham EA stimulation (D) 24 h after operation The mean number of positive reaction cells of c-fos from NTS 6 h (E), 12 h (F) and 24 h (G) after operation Scale bar represent 200 μ m n = 6–8 rats per group and data are means ± SEM **P < 0.01

versus control group

Figure 4 Nucleus of solitary tract neurons excitation after abdomen operation Manipulation of the

gastrointestinal tract and the inhaled anesthetized suppressed the NTS neurons excitation till 24 h after operation and EA excited the NTS The distribution of three types neuron in NTS of four group immediately

(A), 6 h (C), 12 h (E) and 24 h (G) after operation and the rate of change of spike frequency of NTS neurons

10 min (B), 6 h (D), 12 h (F) and 24 h (H) Results of three type neurons’ number are presented as count data

and results of the rate of change of spike frequency are mean, quartile and standard deviation **P < 0.01 versus

control group, ΔΔP < 0.01 versus model group, ##P < 0.01 versus EA group.

Figure 5 EA excitatory effect on nucleus of solitary tract neurons on normal rats EA stimulation excited

the NTS neurons when administrated to normal rats The distribution of three types neuron in NTS of four

group 2 min (A), 4 min (C), 6 min (E), 8 min (G) and 10 min (I) after EA stimulation and the rate of change of spike frequency of NTS neurons 2 min (B), 4 min (D), 6 min (F), 8 min (H) and 10 min (J) Results of three type

neurons’ number are presented as count data and results of rate of change of spike frequency are mean, quartile and standard deviation *P < 0.05 versus normal group.

Figure 6 Leukocyte infiltration in intestinal wall after abdomen operation Manipulation of the

gastrointestinal tract produced a pronounced leukocyte infiltration in intestinal wall from 6 h to 24 h after surgery and EA stimulation had little effect on infiltration MPO staining of muscle whole mounts from rats that

underwent a laparotomy alone (A), a laparotomy with intestinal manipulation (B), an intestinal manipulation with EA stimulation (C) and intestinal manipulation with sham EA stimulation (D) 24 h after operation The mean number of MPO-positive cells in small intestine 6 h (E), 12 h (F) and 24 h (G) after operation Scale bar

represent 20 μ m n = 6–8 rats per group and data are means ± SEM **P < 0.01 versus control group.

Figure 7 IL-1β and TNF-α mRNA expression in small intestinal after abdomen operation Manipulation

of the gastrointestinal tract produced a pronounced IL-1β and TNF-α mRNA expression in small intestinal from 6 h to 24 h after surgery and EA stimulation had little regulation IL-1β mRNA relative expression level

in intestinal wall of four group 6 h (A), 12 h (B) and 24 h (C) after operation TNF-α mRNA relative expression level in intestinal wall of four group 6 h (D), 12 h (E) and 24 h (F) after operation n = 6–8 rats per group and

data are means ± SEM **P < 0.01 versus control group.

Discussion

POI has been characterized by GI dysfunction and a series of clinical symptom, such as abdominal pain and distension, oral diet intolerance, and passage of flatus and stool delay22 Because of its adverse clinical consequences, POI is one of the most important contributors to longer hospital stays and higher costs23 A way to promote recovery of

GI function after abdominal surgery has been desired for decades, but the results have been disappointing11,24

In the present study, we demonstrated that EA stimulated on Zusanli (ST36) improved post-operative intestinal transit, increased the post-operative gastric emptying and shortened the time of post-operative cacation EA promoted NTS excitation after abdominal surgery but did not reduce the inflammatory cell influx or the local inflammation of small intestine in response to IM Our results are line with earlier studies suggesting that EA regulated the GI function via vagus nerve system25 However, our results contrasted with findings that activating the vagus before surgery reduce the inflammation of the small intestine

It is generally believed that three factors suppress GI motility after abdominal surgery26 They are adrenergic and noradrenergic inhibitory neuronal pathways that activated by the nociceptive stimuli during surgery1,27, anesthetics and opioid analgesics used during and after the surgery28–30, and the local inflammatory response to intestinal handling during the surgery5 Anesthetics were once thought to be the main factor leading to POI Now, the local inflammation of the intestine, induced by IM, is considered the main mechanism underlying POI and the most viable therapy target31 The relationship between intestinal inflammation and POI was first reported at the end of the 20th century3,32,33 Then, Kalff first demonstrated the correlation between inflammation and POI in the human body at 200323 Manipulation of the intestine during the abdominal surgery, including laparoscopic surgery34, activates the resident innate immune cells located within the muscularis externa, which initiates the circulating leukocytes invading the small intestinal wall1,26,35,36 The infiltration of leukocytes is not restricted to the location of handling but fast spreads to all areas of the small intestine37 The mechanisms of the inhibition of

GI motility by this local inflammation are not clear yet The leukocytes and resident innate immune cells released various cytokines and chemokines4,36,38, which not only compromised the contractile activity but also excite the inhibitory neural pathways5,6 and inhibit the vagus nerve system affecting the entire gastrointestinal tract39 Here, the transit function of the whole GI tract was significantly suppressed from 6 h to 24 h after surgery, with leuko-cytes infiltrated into the small intestinal muscular layer These results are consistent with previous studies sup-porting the relationship between local inflammatory response and POI Interestingly, the small intestinal motility function of control rats gradually recovered from 6 h to 12 h after operation, which was in line with the previous results showing that anesthetics are involved in the early POI Thus, we believe that the delay of GI transition in

Figure 8 Atropine inhibit EA’s effect EA’s regulatory effect on gastrointestinal transit, colonial transit time

and gastro emptying was inhibited by atropine when it was administrated after operation, but not before

The distribution of orally administered FITC-dextran in whole gastrointestinal tract (A) and the mean GC of fluorescent marker (B) 24 h after surgery The mean colonic transit time (C) and the average gastric retention

24 h after operation (D) n = 6–8 rats per group and data are means ± SEM ΔP < 0.05, ΔΔP < 0.01 versus model

group, &P < 0.05, &&P < 0.01 versus atr + EA group.

study Our results show that EA indeed recovered small intestinal motility function at 24 h after surgery and significantly promoted the colon transit function and gastric emptying All of these results indicate that EA short-ened the recovery time of the stomach, small intestine and colon, as supported by clinical reports54 To learn the mechanisms underlying the treatment effect of EA on POI and further enhance it, we detected the excitation of the NTS, the central nucleus of the vagus reflex, by electrophysiology and c-fos expression to investigate the vaugs nerve excitation

The NTS may be activated after abdominal surgery31, and the brain may sense peripheral inflammation partly through vagal afferents and produce an anti-inflammatory response via vagal efferent fibers55 The intestinal inflammatory response to IM and the NTS activation during POI has been demonstrated15,56 Thus, the vagus nerve system may be activated by the inflammation in small intestine to reduce the inflammatory action via the cholinergic anti-inflammatory pathway In our results, c-fos expression in the NTS was significantly increased after operation at all surgery rats However, the results of electrophysiology were different from the immunofluo-rescence results in this study The spike frequency and population of excited NTS neurons were significantly sup-pressed after surgery In particularly, the exciting of NTS neurons in control rats was supsup-pressed at immediately and 6 h after operation, which was line with the generally belief that anesthesia inhibits central nerve system57 and the result that GI transition was weakened at 6 h after operation in the control group In contrast, a low exciting

of NTS neurons from 6 h to 24 h after operation was consistent with the previous result that abdominal sur-gery excited the NANC pathway inhibit vagus nerve39 Furthermore, the control rats’ NTS exciting was strongly excited from 6 h to 24 h, which may be induced by hunger However, there were little c-fos expression in NTS in control rats We think that NTS, as a parasympathetic center of the vago-vagal reflex, detected the inflammatory response at the intestinal wall but some factors, such as anesthesia and inhibitory neurons, might block its excita-tion The relationship between c-fos expression and the neuronal exciting electric signaling will be studied in our further research

When investigated the EA’s effect on NTS, we observed that EA stimulation did not regulate the c-fos expres-sion in the NTS at any time point However, EA significantly increased the NTS exciting from 6 h to 24 h after surgery Unfortunately, we did not observe any effect of EA on local inflammation The leukocyte invasion into the intestinal wall from 6 h to 24 h after surgery and peaked at 12 h after operation Although EA activated the NTS at 6 h, 12 h and 24 h after operation, it did not decrease the level of leukocyte infiltration Activation of the resident macrophages is the first step in leukocyte infiltration of the small intestine26,58 Hence, vagus stimu-lation prevented the local inflammatory response via inhibition of macrophage activation by the cholinergic anti-inflammatory pathway Our results show the excitatory effect of EA on NTS in normal rats, which was con-sistent with previous studies25,52 However, the NTS excitation effect of EA was detected at 6 h, not immediately after surgery These results indicate that the effect of EA, administered before and during the surgery on NTS was interfered with some factors EA is generally considered to excite the NTS via somatic afferents, then activated the vagus efferent fibers, inducing acetylcholine release So EA’s effect may have been disturbed by the anesthetic effect of isoflurane, whether on NTS or the peripheral nerve system However, the cholinergic anti-inflammatory pathway mainly affects macrophages, not leukocyte When leukocyte had invaded the intestinal wall, EA admin-istered at 6 h after operation failed to prevent inflammation All the above are the reason why we think EA indeed excited the NTS but failed to prevent inflammation Because EA failed to regulate the inflammatory response, we did not further investigate the effect of EA on muscle inhibitory substances

Finally, we combine EA treatment with an intraperitoneal injection of atropine before or after abdominal surgery, to investigate the main mechanism underlying EA’s effect on POI The results indicate that suppressing the vagus nerve system before operation, which also blocked the cholinergic anti-inflammatory pathway, did not affect the regulatory effect of EA on POI In contrast, atropine significantly blocked the regulatory effect of EA

on POI when injected after operation We believed that the therapeutic effect of EA on POI mainly depended on the NTS-promoting effect in the GI tract, which was induced by EA administered after operation, but not the anti-inflammation effect of vagus excitation that might be produce by preoperative EA

We showed that EA reduced the recovery time of POI after abdominal surgery This effect is mainly mediated

by the exciting of the NTS to improve GI transit function However, we failed to prevent the local inflammation

of the small intestine by the vague excitation effect of EA treatment Further exploration of EA administration methods that activate the cholinergic anti-inflammatory pathway after abdominal operation may improve the treatment effect of EA on POI and are the subject of ongoing investigation

Methods and Materials

Animals and groups All rats were obtained from the animal experiment center attached to Zhejiang Chinese Medical University Rats used in electrophysiological experiments weighted 280–300 g, and others were 200–220 g They were housed with an artificial 12-h light-dark cycle at a controlled temperature (23 ± 1 °C) and relative humidity (70 ± 10%) Distilled water and food were available ad libitum Rats were fasted from 12 h before surgery to 24 h after surgery with water available ad libitum All animal care, surgery, and handling procedures were approved by the animal experiment center attached to Zhejiang Chinese Medical University and performed strictly in accordance with the National Institutions of Health Guide for the Care and Use of Laboratory Animals

Surgical procedures POI was induced by a standardized small bowel manipulation procedure7 Rats were anesthetized with inhaled isoflurane, and the abdomen was opened by midline laparotomy The small intestine was carefully exteriorized and then gently manipulated along its entire length using moistened sterile cotton applicators Finally, the bowel was repositioned in the abdominal cavity, and the incision was closed by a contin-uous 2-layer suture The rats were allowed to recover in the same cage

Electrode implantation surgery Rats were deeply anesthetized with urethane (1 g/kg, i.p.) and trans-ferred to a stereotaxic instrument Craniotomy was made for microelectrode array implantation in one side of the brain According to the atlas of Paxinos and Watson (Edition VI), the NTS was located as follows: 12.8 mm pos-terior to bregma, 0.8 mm lateral to the midline, and 5.8 mm ventral to the skull surface An array of eight stainless steel Teflon-insulated microwires (50μ m thick) were slowly lowered into the NTS The microelectrode arrays were secured onto the cranium with stainless steel skull screws and dental cement Rats were administered penicillin (20,000 U, i.m.) and allowed 7 days to recover

EA and sham EA stimulation During the whole EA stimulation, rats were loosely immobilized by an assistant’s hands, except during the surgical process Four stainless steel acupuncture needles 0.25 mm in diame-ter were inserted at a depth of 5 mm into the biladiame-teral “Zusanli” (ST36, between the tibia and fibula 5 mm below the knee) acupoints and reference points (1 cm below ST 36) The two ipsilateral needles were connected to the output terminals of the HANS Acupuncture point Nerve Stimulator (LH-202H, Huawei Co., Ltd., Beijing, China) Electro-stimulation was delivered with constant parameters, constant square wave current output at 5 Hz (pulse width: 0.2 ms), and intensities ranging from 1 to 2 mA (each intensity for 15 min, totaling 30 min) The sham EA group received the same subcutaneous needle insertion (2 mm in depth) into ST36 and reference points, and the needles were linked to the electrodes but without electrical stimulation To eliminate the stress effect, rats in the model and sham EA groups were loosely immobilized by an assistant’s hands similar to the EA group

Gastrointestinal transit To determine the effects of IM and EA on gut motility, GI transit was meas-ured at 6 h, 12 h and 24 h after IM as previously described7 Two hundred microliters of a liquid non-absorbable FITC-dextran (70,000 Da, Sigma-Aldrich, USA) was administered via oral gavage Forty minutes later, rats were sacrificed, and the entire gastrointestinal tract from stomach to distal colon was collected The contents of the stomach, small bowel (equal divided into 10 segments), cecum, and colon (equal divided into 3 segments) were flushed and collected Finally, the fluorescent signal in each bowel segment was quantified in duplicate (excitation wavelength: 485 nm, emission wavelength: 528 nm) by a microplate reader (Spectra Max M4, Molecular Devices, USA) For statistical analysis, a geometric center (GC) was calculated to represent the GI function: Σ (% of total fluorescent signal in each segment × the segment number)/100

Colonic transit Colonic function was investigated in vivo 6 h, 12 h and 24 h after IM by measuring the colon

transit time of a stainless steel ball A 5 mm diameter stainless steel ball was carefully inserted 4.5 cm into the colon with a polished metal rod via the anus The time from insertion until excretion of the ball was considered the colonic transit time

Gastric emptying The gastric emptying was measured at 24 h after IM by a semi-liquid, non-caloric test meal (2 mL of 1.5% methylcellulose solution containing 0.1% phenol red) Animals were sacrificed 40 min after oral gavage and the stomach was immediately removed with gastro-esophageal and gastroduodenal clamping The stomach was rinsed with phosphate-buffered saline (PBS, pH 7.4) and dissected in 18 mL normal saline (NS) Twenty milliliters of 0.5 M NaOH was added to the NS, and the contents of the stomach, containing phenol red solution, were mixed Five milliliters of supernatant was collected, and 0.5 mL of 20% trichloroacetic acid was added to precipitate proteins The samples were centrifuged at 3600 rpm for 10 min, and the absorbance of sam-ples at 560 nm wavelength was measure with a spectrophotometer (Spectra Max M4, Molecular Devices, USA)

A control sample was prepared as follow: 2 mL 1.5% methylcellulose solution containing 0.1% phenol red, 18 mL

NS, 20 mL 0.5 M NaOH and 4 mL 20% trichloroacetic acid The absorbance of the control sample at 560 nm wave-length was also measured The gastric emptying was estimated from the following formulation: (1 − absorbance

of residual phenol red in stomach absorbance of control phenol red) × 100%

In vivo electrophysiological recording The neuronal activities were investigated before and after the EA stimulation During the recording session, rats were moving freely The neuronal activities were detected by the microwires and passed from the headstage to a preamplifier Single activities were recorded using a 128-channel data acquisition system (Cerebus, Blackrock Microsystems, USA) The neural signals were analog-filtered by the amplifier at cutoff frequencies of 0.3 Hz and 7.5 kHz and digitized at a 16-bit resolution at 30,000 Hz using a Cerebus Neural Signal Processor The digitized signals from each microwire were amplified and digitized band-pass filtered from 250 Hz to 5000 Hz Finally, all the signals were saved into a data file for off-line analysis Spike activities were extracted from the digitized recordings, and individual units were isolated offline using Plexon